Spasmolytic effect of stachys lavandulifolia vahl. Crude methanolic extract and fractions on rat ileum.

The aim of this study was to investigate the effect of aerial parts of Stachys lavandulifolia Vahl. Extract on the rat ileum contractions. The crude extract was prepared by maceration method (90% methanol) followed by fractionating into chloroform, ethyl acetate, petroleum ether and water. In adult male Wistar rats, ileum was sectioned and mounted in tissue bath and their contractility was recorded is otonically. KCl (60 mM)- induced ileum contractions were attenuated by crude extract and its fractions. The most potent fraction was chloroformic fraction (CF) with IC50 0.018 ± 0.126 = mg/mL. In calcium-free Tyrode solution with high K(+,) the CF (0.01 - 0.04 mg/ml) attenuated CaCl2-induced contractions (p< 0.001). The CF (0.05-0.8 mg/mL) attenuated the carbachol-induced contraction (p<0.001). The CF antispasmodic effect was reduced by naloxone (as a non-selective opioid antagonist), not by propranolol and L-NAME as β-adrenoceptors antagonist and nitric oxide synthase inhibitor respectively. It was concluded that S . . lavandulifolia can inhibit ileum contractility mainly via disturbing the calcium mobilization and partly by opioid receptors' activation. Our results may support the traditional usage of this herb for treating diarrhea.


Introduction
Diarrhea continues to be one of the leading causes of mortality and morbidity especially in children in developing countries (1). Diarrhea is characterized by hypermotility of gastrointestinal tract (2). Natural products have served as a medicinal source for centuries, and about half of the pharmaceuticals in use today are derived from natural products (3). Stachys lavandulifolia Iranian traditional medicine as an anxiolytic and to treat diarrhea (4). Furthermore, experimental studies have demonstrated the anxiolytic effect of S. lavandulifolia f in mice (5, 6). Despite the extraction of S. lavandulifolia constituents such as -pinene, myrcene, -phellandrene and -caryophyllene (4), its pharmacological effects on isolated smooth muscle have not aim of the present study was to investigate the antispasmodic potential of the S. lavandulifolia crude extract and its fractions on rat ileum.

Experimental
Plant material Stachys lavandulifolia Vahl. was collected (March 2008) from Zanjan province (center of Iran) and authenticated by Dr. Namjoyan from the Pharmacognosy Department of the Ahwaz Jundishapur University of Medical Sciences (AJUMS). A voucher specimen was deposited in the herbarium of the School of Pharmacy of AJUMS.

Extraction and fractionation
The aerial parts of the herb were dried in shade. The powder of the herb was macerated with methanol (90%) for 72 h at room temperature to obtain the crude extract (CE).
evaporator and was air-dried in the next step (yield: 17.22%). Petroleum ether fraction (PF) was prepared from the CE (16.8 g) being petroleum ether. The extraction was reported for three times followed by concentrating and air-drying (yield: 14.27%). In order to obtain the chloroformic fraction (CF), the PF was mixture was concentrated and air-dried (yield: 1.74%). The same procedure was repeated in order to obtain ethyl acetate fraction (EF) using was reported for three times and the mixture was concentrated and thereafter dried (yield: 5.23%). The remaining aqueous fraction (AF) was air-dried (yield: 77%). The extract and fractions were stored at 4 º C until being used.

Animals
Male Wistar rats (190 ± 10 g) were purchased from Animal Facility of AJUMS, housed at 20-24 C with 12 h/12 h light/dark cycle and free access to food and water. All animals used in this study were treated in accordance with principals and guidelines on Ethics Committee of AJUMS. The rats were deprived of food (not water) for 24 h before the experiment.

Ileum preparation
by a sharp blow on the head and one or two pieces (1.5-2 cm) of ileum were dissected out from 2 cm above the ileocaecal junction and mounted between two stainless steel hooks in tissue bath C and bottom of the tissue bath and the upper one was connected to an isotonic transducer (Harvard Transducer, UK). Each tissue was placed under 1 g resting tension and equilibrated for 60 min prior to the execution of experimental protocols. During this period, the tissue was washed with Tyrode solution every 15 min and the tension was readjusted to 1 g. Ileum contractions were displayed and recorded on Universal Harvard Oscillograph, (UK). Tyrode solution composition consisted of (in mM) NaCl (136.9), KCl (2.6), CaCl 2 (1.8), NaHCO 3 (11.9), NaH 2 PO 4 (0.42), MgCl 2 (1.05) and glucose (5.55) which continuously was bubbled with air. The ileum contractions were induced by KCl (60 mM) or was achieved, the crude extracts or fractions bath. The mechanism of CF spasmolytic activity was studied by separate tissue incubations either 30 min) or N G -adrenoceptors, opioid receptors antagonists and nitric oxide synthase inhibitor, respectively. evaluated by depolarizing the ileum in Ca 2+ -free Tyrode solution with high K + (60 mM) and then applying CaCl 2 (0.2-0.6 mM) cumulatively to induce contraction. Same protocol was repeated after incubating the ileum with CF (0.01-0.04 Chemicals purchased from Sigma (Sigma, USA), naloxone was purchased from Tolidaru Company (Tolidaru, Iran) and DMSO was purchased from Merck (Merck, Germany).

Data analysis
Plateau of ileum KCl-or CCh-induced contraction was assumed as 100% and the relaxations evoked by extract or fractions, were calculated (as %) and the results were expressed as mean ± SEM for n number of animals. Multiple means were compared by one-way and two-way analysis of variance (ANOVA) followed by Dunnett>s test. A p-value of less than 0.05 was analysis was performed using SPSS (version 16.0).

Results and Discussion
Antispasmodic activity of the CE Cumulative concentrations of CE (0.125-4 ileum contraction (IC 50 in a concentration-dependent manner (Figure 1).

Effects of L-NAME, propranolol and naloxone on CF antispasmodic activity
In the ileum precontracted by KCl, the chloroformic fraction (two-way ANOVA, p < 0.01) (Figure 3).

Effect of CF on CaCl 2 l -induced ileum contraction
In Ca 2+ -free with high K + (60 mM) Tyrode solution, cumulative concentrations of CaCl 2 (0.2-0.6 mM) induced ileum contractions in a concentration-dependent manner (one-way ANOVA, p < 0.001). Five min ileum incubation CaCl 2 -induced contractions. The CaCl 2 -induced ileum contractions in the absence and in the presence of lowest concentration of CF (0.01 ANOVA, p < 0.001) (Figure 4).

Effect of CF on CCh-induced ileum contraction
contraction in a concentration-dependent fashion (one-way ANOVA, p < 0.001) and IC 50 was Chloroformic fraction (n = 8) was more potent than PF and EF (two-way ANOVA, p < 0.001). The spasmolytic effects of PF (n = 7) and EF (n = 10) were not different and AF (n = 7) was ineffective.

activities on contractions induced by KCl and
CCh have been compared in Figure 5.

Effect of DMSO on KCl and CCh-induced ileum contractions
Since DMSO was used to solve S. lavandulifolia fractions, the possible role of DMSO in the CF antispasmodic activity was studied by applying this solvent to the ileum contractions induced by KCl (60 mM) or CCh were unaffected by DMSO (0.005-0.08 w/w).

Discussion
study was the antispasmodic effect of S. lavandulifolia on rat ileum. The observed spasmolytic effect was not reversible completely since the ileum responsiveness to spasmogens was not reestablished after refreshing the tissue bath solution. The comparison of spasmolytic effect of crude extract and fractions indicated that chloroformic fraction (CF) with lowest IC 50 was the most potent fraction.
The contraction in the smooth muscle is Ca 2+ which can be provided by two main voltage-dependent calcium channels; 2) by releasing Ca 2+ from intracellular calcium pools such as sarcoplasmic reticulum (7). In the KClinduced smooth muscle contraction, the voltagedependent calcium channels (VDCCs) are in rat ileum has been reported (9). On the other hand, CCh (muscarinic receptor agonist) induces ileum contraction via M 2 and M 3 receptors (10). Binding CCh to these receptors increases intracellular calcium concentration (11) by both Ca 2+ 2+ channels and releasing calcium from cytoplasmic calcium stores (8). The ileum contractions induced by the KCl-induced contraction but relatively inhibited (66%) the CCh-induced contraction. Thus, the KCl-induced contraction was more susceptible to CF than the one elicited by CCh, which suggested that events at the cholinergic receptors level were not important.
It has been suggested that the substance that inhibits the KCl-induced smooth muscle contraction is considered as a Ca 2+ (12). The result of CF spasmolytic effect on CaCl 2induced contractions supports this suggestion, since high K + in the Ca 2+ -free Tyrode solution depolarizes the ileum (13) and the contraction may occur only after applying CaCl 2 (11). Furthermore, KCl and CCh increase intracellular

CCh + CF
Ca 2+ by activating the calcium channels which suggests that CF of S. lavandulifolia has inhibited Ca 2+ mobilization.
Activation of f f -adrenoceptor activation evokes the ileum relaxation (14). The ineffectiveness of propranolol in attenuating the CF spasmolytic effect, however, indicates that -adrenoceptors were not involved in CF activity.
Nitric oxide as a main inhibitory neurotransmitter in the gut (15) modulates the contractility of small intestine through cGMP oxide synthase inhibitor) to attenuate the CF spasmolytic effect indicates that CF activity was not mediated through nitric oxide. Opioid receptors activation relaxes the ileum smooth muscle (17) and in our study, naloxone (nonselective opioid receptors antagonist) partially suggests that CF activity has been mediated, at least partially, via these receptors. Furthermore, the KCl-and CCh-induced ileum contractions were unaffected by DMSO indicating that the observed CF spasmolytic activity was not due to DMSO effect.
The main constituents of S. lavandulifolia are alpha-pinene, myrcene, -phellandrene and -caryophyllene (4) which some of them have spasmolytic effect (18,19). The existence of apigenin and luteolin in S. lavandulifolia (20) and the antispasmodic effects of these two therefore, it seems that the observed spasmolytic effect of S. lavandulifolia was due to its 50 of the chloroformic fraction of S. lavandulifolia on which, however, further research is needed to In conclusion, S. lavandulifolia Vahl. chloroformic fraction of aerial parts, particularly, induced spasmolytic effect in the isolated rat and partly via opioid receptors activation. The obtained results support the traditional usage of this herb to treat diarrhea.